The use of genetic recombination technology makes it possible to impart new traits to plants by expressing a useful gene in a target plant. A wide range of genetically modified plants produced in this manner have already been cultivated. Since regulation of gene expression is mainly controlled at the level of transcription, transcriptional regulation is the most important in terms of regulating the expression of genes. Namely, transcribing a gene at a suitable time, in a suitable tissue and at a suitable strength is important for producing an industrially useful genetically modified plant. In many cases, initiation of transcription is controlled by a DNA sequence on the 5′-side of a translated region, while termination is controlled by a DNA sequence on the 3′-side of a transcribed region. A region of DNA that determines the starting site of gene transcription and directly regulates the frequency thereof is referred to as a promoter, while the region that determines termination of transcription is referred to as a terminator. A promoter is located several tens of base pairs (bp) from the 5′-side of an initiation codon, and frequently contains a TATA box and the like. A cis element that binds various transcriptional regulatory factors is also present on the 5′-side, and the presence thereof serves to control the timing of transcription, the tissue in which transcription takes place and transcriptional strength. Transcriptional regulatory factors are classified into many families according to their amino acid sequence. For example, examples of well-known families of transcriptional regulatory factors include Myb type transcriptional regulatory factors and bHLH (basic helix loop helix) type transcriptional regulatory factors. In actuality, the terms transcriptional regulatory factor and promoter are frequently used with the same meaning and are not strictly distinguished.
Anthocyanins, which compose the main components of flower color, are a member of secondary metabolites generically referred to as flavonoids. The color of anthocyanins is dependent on their structure. Namely, color becomes blue as the number of hydroxyl groups of the B ring of anthocyanidins, which is the chromophores of anthocyanins, increases. In addition, as the number of aromatic acyl groups (such as coumaroyl groups or caffeolyl groups) that modify the anthocyanin increases, the color of the anthocyanin becomes blue (namely, the wavelength of maximum absorbance shifts to a longer wavelength) and the stability of the anthocyanin is known to increase (see Non-Patent Document 1).
Considerable research has been conducted on those enzymes and genes that encode those enzymes involved in the biosynthesis of anthocyanins (see, Non-Patent Document 1). For example, an enzyme gene that catalyzes a reaction by which an aromatic acyl group is transferred to anthocyanin is obtained from Japanese gentian, lavender and petunias (see Patent Document 1 and Patent Document 2). Several enzyme genes involved in the synthesis of anthocyanin that accumulates in the leaves of perilla (malonylcyanin, 3-O-(6-O-(E)-p-coumaroyl-β-D-glucopyranosyl)-5-O-(6-O-malonyl-β-D-glucopyranosyl)-cyanidin) (see Non-Patent Document 2) have previously been reported, including human hydroxycinnamoyl CoA: anthocyanin-3-glucoside-aromatic acyl transferase (3AT) gene (or more simply referred to as “perilla anthocyanin-3-acyl transferase (3AT) gene”) (see Patent Document 1). Moreover, findings have also been obtained regarding the transcriptional regulation of biosynthetic genes of anthocyanins. Cis element sequences bound by Myb type transcriptional regulatory factor and bHLH type transcriptional regulatory factor are present in the transcriptional regulatory region located on the 5′-side of the initiation codons of these genes. Myb type transcriptional regulatory factor and bHLH type transcriptional regulatory factor are known to control synthesis of anthocyanins in petunia, maize and perilla (see Non-Patent Document 1).
Promoters (also referred to as transcriptional regulatory regions) responsible for gene transcription in plants consist of so-called constitutive promoters, which function in any tissue and at any time such as in the developmental stage, organ/tissue-specific promoters, which only function in specific organs and tissues, and time-specific promoters, which only express genes at a specific time in the developmental stage. Constitutive promoters are frequently used as promoters for expressing useful genes in genetically modified plants. Typical examples of constitutive promoters include cauliflower mosaic virus 35S promoter (to also be abbreviated as CaMV35S) and promoters constructed on the basis thereof (see Non-Patent Document 3), and Mad promoter (see Non-Patent Document 4). In plants, however, many genes are only expressed in specific tissues or organs or are only expressed at specific times. This suggests that tissue/organ-specific or time-specific expression of genes is necessary for plants. There are examples of genetic recombination of plants that utilize such tissue/organ-specific or time-specific transcriptional regulatory regions. For example, there are examples of protein being accumulated in seeds by using a seed-specific transcriptional regulatory region.
However, although plants produce flowers of various colors, there are few species capable of producing flowers of all colors due to genetic restrictions on that species. For example, there are no varieties of rose or carnation in nature that are capable of producing blue or purple flowers. This is because roses and carnations lack the flavonoid 3′,5′-hydroxylase (hereinafter simply referred to as F3′5′H) gene required to synthesize the anthocyanin, delphinidin, which is synthesized by many species that produce blue and purple flowers. These species can be made to produce blue flowers by transforming with the F3′5′H gene of petunia or pansy, for example, which are species capable of producing blue and purple flowers. In this case, the transcriptional regulatory region of chalcone synthase gene derived from snapdragon or petunia is used to transcribe F3′5′H gene derived from a different species. Examples of plasmids containing the transcriptional regulatory region of chalcone synthase gene derived from snapdragon or petunia include plasmids pCGP485 and pCGP653 described in Patent Document 3, and examples of plasmids containing a constitutive transcriptional regulatory region include plasmid PCGP628 (containing a Mad promoter) and plasmid pSPB130 (containing a CaMV35S promoter to which is added EI2 enhancer) described in Patent Document 4.
However, it is difficult to predict how strongly such promoters function in recombinant plants to be able to bring about a target phenotype. In addition, transforming a plant with the same or similar base sequence, creating numerous copies of a introduced gene in chromosomes or repeatedly inserting a transgene may cause gene silencing (see Non-Patent Document 5). Thus, since repeatedly using the same promoter to express a plurality of exogenous genes may cause gene silencing, this should be avoided.
On the basis of the above, although several promoters have been used to alter flower color, a promoter is still required that is useful for changing to other flower colors corresponding to the host plant.